Department of Biomedical Sciences, University of Barcelona, Barcelona 08036, Spain.

Abstract

Persistent activation of the innate immune system greatly influences the risk for developing metabolic complications associated with obesity. In this study, we explored the therapeutic potential of the specialized proresolving mediator (SPM) resolvin D1 (RvD1) to actively promote the resolution of inflammation in human visceral adipose tissue from obese (Ob) patients. Using liquid chromatography-tandem mass spectrometry-based metabololipidomic analysis, we identified unbalanced production of SPMs (i.e., D- and E-series resolvins, protectin D1, maresin 1, and lipoxins) with respect to inflammatory lipid mediators (i.e., leukotriene B4 and PGs) in omental adipose tissue from Ob patients. In parallel, high-throughput transcriptomic analysis revealed a unique signature in this tissue that was characterized by overactivation of the IL-10 signaling pathway. Incubation of inflamed Ob visceral adipose tissues and human macrophages with RvD1 limited excessive activation of the IL-10 pathway by reducing phosphorylation of STAT proteins. Of interest, RvD1 blocked STAT-1 and its target inflammatory genes (i.e., CXCL9), as well as persistent STAT3 activation, without affecting the IL-10 anti-inflammatory response characterized by inhibition of IL-6, IL-1β, IL-8, and TNF-α. Furthermore, RvD1 promoted resolution by enhancing expression of the IL-10 target gene heme oxygenase-1 by mechanisms dependent on p38 MAPK activity. Together, our data show that RvD1 can tailor the quantitative and qualitative responses of human inflamed adipose tissue to IL-10 and provide a mechanistic basis for the immunoresolving actions of RvD1 in this tissue. These findings may have potential therapeutic implications in obesity-related insulin resistance and other metabolic complications.

(A) Representative immunoblot of STAT3 and phospho-STAT3 (pSTAT3, phosphorylation at Tyr705, pSTAT3Tyr) assessed by western blot in visceral adipose tissue samples from control (CT, n=8) and obese (Ob, n=14) individuals. The densitometric analysis of pSTAT3Tyr to total STAT3 ratio is shown on the right. (B) Representative immunoblots of pSTAT3Tyr and total STAT3 levels in adipose tissue explants incubated with increasing concentrations of RvD1 (0, 1, 10 and 50 nM) for 30 min followed by the addition of IL-10 (20 ng/ml) for 30 or 120 min. (C) Kinetic analysis of STAT3Tyr phosphorylation in adipose tissue explants incubated in the absence (solid circles) or presence (empty circles) of RvD1 and stimulated with IL-10. (D) Representative immunoblots analyzing STAT3 activation in THP1 monocytes incubated in the presence of increasing concentrations of IL-10 for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. (E) Representative immunoblots of STAT3 and pSTAT3Tyr in THP-1-derived macrophages incubated in the presence of increasing concentrations of RvD1 for 30 min followed by the addition of IL-10 (20 ng/ml) for 10 min. Densitometric analysis of phosphorylated protein to total protein ratios is shown below. Results are expressed as mean ± SEM of n=3 independent experiments performed in duplicate. P values are given vs CT. *, P<0.05 vs vehicle (0.01% ethanol) at time 0. a, P<0.05 and b, P<0.01 for RvD1 vs vehicle at time 120 min.

(A) Representative immunoblots of STAT3 and STAT3 Tyr705 phosphorylation (pSTAT3Tyr) assessed by western blot in adipose tissue explants incubated with increasing concentrations of RvD1 (0, 1, 10 and 50 nM) for 30 min followed by the addition of IL-6 (10 ng/ml) for 30 or 120 min. (B) SOCS3 mRNA expression in adipose tissue from control (CT, n=8) and obese (Ob, n=24) individuals. (C) mRNA expression for SOCS3 and SOCS1 was analyzed in adipocytes (ADP) and stromal vascular cells (SVC) isolated from human obese adipose tissue. (D) Representative immunoblot of STAT3 and pSTAT3Tyr in THP-1 monocytes incubated in the absence (−) or presence (+) of RvD1 (10 nM) for 30 min followed by the addition of increasing concentrations of IL-6 for 10 min. (E) Representative immunoblots of SOCS3 and GAPDH in THP-1 cells pre-incubated with vehicle (0.2% ethanol) or MG132 (10 μM) for 30 min before the addition of IL-6 (10 ng/ml) for 5 h. (F) Representative immunoblots of STAT3 and pSTAT3Tyr in THP-1 cells pre-incubated with IL-10 (20 ng/ml) for 30 min before the addition of IL-6 (10 ng/ml) in the presence of vehicle (0.2% ethanol) or MG132 (10 mM) for 5 h. (G) Relative mRNA levels for both IL-6 signal transducer (ST), also known as gp130, and IL-6 receptor (R) genes in human adipose tissue from CT (n=6) and Ob (n=18) subjects were assessed by real-time PCR relative to a calibrator sample consisting of a total RNA derived from normal human adipose tissue pooled from 18 individuals. (H) Representative inmmunoblots of STAT3 activation (left panel) assessed by western blot in adipose tissue explants incubated with increasing concentrations of RvD1 (0, 1, 10 and 50 nM) for 30 min followed by the addition of IL-1β (25 pg/ml) for 30 or 120 min and kinetic analysis of STAT3Tyr phosphorylation (right panel) in adipose tissue explants incubated in the absence (solid circles) or presence (empty circles) of RvD1 (10 mM) and stimulated with IL-1β. (I) Representative immunoblots of STAT3 and pSTAT3Tyr in THP-1-derived macrophages incubated in the absence (−) or presence (+) of RvD1 (10 nM) for 30 min followed by the addition of either IL-1β (25 pg/ml) or IL-6 (10 ng/ml) for 10 min. Results are expressed as mean ± SEM of n=3 independent experiments performed in duplicate. P values are given vs CT or ADP. *, P<0.05 and **, P<0.005 vs vehicle (0.01% ethanol) at time 0.

Schematic diagram of the interaction of RvD1 with the IL-10 signaling pathway

IL-10 promotes a STAT3-mediated anti-inflammatory response characterized by reduced expression of pro-inflammatory cytokines and increased expression of anti-inflammatory targets. Persistent activation of STAT signaling by IL-10 results in a pro-inflammatory response characterized by the up-regulation of STAT1-dependent CXCL9 target. In these conditions, RvD1 limits excessive overactivation of the STAT pathway while maintaining the IL-10-induced anti-inflammatory response via positive regulation of HO-1 through the p38 MAPK pathway. The p38 MAPK pathway also signals the actions of RvD1 on IL-1ra, a natural inhibitor of the pro-inflammatory actions of IL-1β.